Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus

Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus protei ns in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immune-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.